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  • 1
    Electronic Resource
    Electronic Resource
    Characteristics of immunofluorescence microscopy and of dilution-plating to detect Pseudomonas syringae pv. phaseolicola in bean seed lots and for risk assessment of field incidence of halo blight (1991)
    Springer
    European journal of plant pathology 97 (1991), S. 233-244 
    Details
    ISSN: 1573-8469
    Keywords: Phaseolus vulgaris ; Phaseolus coccineus ; bush bean ; runner bean ; method evaluation ; grey test area ; detection level ; saprophytic interference ; predictive value ; diagnostic sensitivity ; diagnostic specificity ; analytical sensitivity ; analytical specificity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Routine laboratory testing of 710 bean seed lots from various origins forPseudomonas syringae pv. phaseolicola (Psp) with immunofluorescence microscopy (IF) showed that 27.5% of the seed lots (five subsamples of 1000 seeds tested per sample) contained two or more IF-positive cells in a total of 500 microscope fields (magnification 500×). Simultaneously performed dilution-platings of IF-positive subsamples on King's medium B confirmed presence of Psp for one-third of these IF-positive seed lots. The ‘grey area’ of disagreement between both laboratory tests was studied by comparison of test data and by field trials. The number of IF-positive cells per subsample was positively correlated with isolation and identification of Psp (R=0.85). The detection level of IF was ca. 102 Psp cells per ml of undiluted subsample extract. The detection level of Psp by isolation on King's medium B was variable, being inversely related with the saprophyte to Psp ratio. The high sensitivity of IF was in part due to high percentages of dormant or dead IF-positive cells in the sample extract. Field trials over two years with 10 000 seeds per seed lot, showed disease incidence for 9 of the 22 seed lots. Of ten IF-positive lots with five positive subsamples per sample, nine were positive in the field test plot (the negative lot gave primary infection spots of Psp when used for commercial growing). By isolation, seven of these ten IF-positive lots were positive. Of the five IF-positive lots with two or less positive subsamples, isolation and field trial were both negative. Based on data on seed transmission from literature, field incidence was unlikely for these five samples in a 10 000 seeds field trial. All seven IF-negative lots were negative in the field trial. The value of IF and isolation for indexing bean seed lots for Psp is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01989820
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  • 2
    Electronic Resource
    Electronic Resource
    Analysis of the toxicity of purothionins and hordothionins for plant pathogenic bacteria (1993)
    Springer
    European journal of plant pathology 99 (1993), S. 259-268 
    Details
    ISSN: 1573-8469
    Keywords: antibacterial ; antimicrobial ; genetic engineering ; thionin ; toxicity assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Purothionins (PTHs) and hordothionins (HTHs) were purified by cation-exchange chromatography from petroleum-ether extracts of wheat and barley flour respectively. The HTHs could be separated into two fractions, HTH-1 and HTH-2. Radial diffusion assays and micro-plate broth dilution assays with a number of plant pathogenic bacteria showed that these proteins were toxic forClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker on tomato,C. m. subsp.sepedonicus, the causal agent of ring rot on potato, andXanthomonas campestris pv.vesicatoria, the causal agent of a spot disease on tomato and pepper. Only minor differences in toxicity between PTHs and HTHs, and between HTH-1 and HTH-2, were detected. Minor differences in toxicity of these thionins were also detected for different strains of these bacteria. The use of these plant proteins for engineering bacterial disease resistance into solanaceous crops will be discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01974307
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  • 3
    Electronic Resource
    Electronic Resource
    Comparison of immunofluorescence colony-staining in media, selective isolation on pectate medium, ELISA and immunofluorescence cell staining for detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in cattle manure slurry (1990)
    Springer
    European journal of plant pathology 96 (1990), S. 75-89 
    Details
    ISSN: 1573-8469
    Keywords: sample preservation ; sample filtration ; detection level ; total counts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Description / Table of Contents: Samenvatting Verschillende isolatie- en serologische methoden werden vergeleken om de doelbacteriënErwinia carotovora subsp.atroseptica (Eca) enE. chrysanthemi (Ech) aan te tonen in runderdrijfmest met een natuurlijke bacterieflora van ca 108 kolonievormende eenheden(cfu) per ml. De mestmonsters konden gedurende de onderzoekperiode tenminste 8 maanden bij −80°C worden bewaard zonder dat er een afname van het aantal levende mestbacteriën werd geconstateerd, terwijl bij bewaring bij −20°C wel een afname werd gevonden. De ontdooide mestmonsters werden geïnoculeerd met de doelbacterie in concentraties tussen 102 en 108 per ml. De laagste concentratie van de doelbacterie, 102 cfu per ml, kon alleen worden aangetoond met de immunofluorescentie-kleuring van bacteriekolonies (IFC) in een selectief medium. Met deze techniek was het percentage herisolatie vanuit drijfmest geïnoculeerd met 102 Ech cfu per ml respectievelijk 64% in PT-medium (bevat polygalacturonzuur) en 19% in kristalviolet pectine medium (CVP). Voor Eca bedroegen deze percentages respectievelijk 82% en 32%. In de niet-geïnoculeerde mestmonsters werden geen IFC-positieve kolonies gevonden. Via isolatie op CVP konden 103 of meer cfu van Eca en Ech worden aangetoond. Ruwe filtratie van de mestmonsters was nodig voor het aantonen van Eca- en Ech-cellen met immunoadsorptie immunofluorescentie microscopie. De detectiedrempel lag voor deze techniek op 105 bacteriecellen per ml mestmonster. In niet-geïnoculeerde mest werden incidenteel IF-positieve bacteriën gevonden. Het aantonen van Ech en Eca met ELISA was slechts mogelijk in mest geïnoculeerd met 108 of meer cellen van de doelbacterie per ml.
    Notes: Abstract Various isolation and serological techniques were compared for the detection ofErwinia carotovora subsp.atroseptica (Eca) andE. chrysanthemi (Ech) in cattle manure slurry containing c. 108 colony forming units (cfu) per ml. The slurry samples could be preserved at −80°C for 8 months without reduction in the number of bacteria but not at −20°C. Samples stored at −80°C were inoculated with concentrations of the target bacterium ranging from 102 to 108 per ml. Only immunofluorescence colony-staining (IFC) in combination with selective media was able to detect the target organism at a concentration of 100 cells per ml. No IFC-positive colonies were found in pour plates of the non-inoculated cattle slurry. The recovery of the target bacterium from slurry inoculated with 102 cfu of Ech per ml was 64% in PT medium (containing polygalacturonic acid) and 19% in crystal violet pectate medium (CVP). Recoveries of Eca were 32% and 82%, respectively. Ech and Eca could be detected at levels of 103 cfu per ml of slurry by isolation on CVP. Crude filtration procedures were necessary for analysis of slurry samples with immunosorbent immunofluorescence (ISIF) cell staining. The detection level of ISIF for Ech was 105 cells per ml of slurry. IF-positive cells were incidentally observed in the non-inoculated slurry. Detection of Ech and Eca with ELISA was only possible in slurry inoculated with 108 cells of the target bacterium per ml.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02005131
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  • 4
    Electronic Resource
    Electronic Resource
    Development of a semi-selective medium and an immunofluorescence colony-staining procedure for the detection of Clavibacter michiganensis subsp. sepedonicus in cattle manure slurry (1991)
    Springer
    European journal of plant pathology 97 (1991), S. 321-334 
    Details
    ISSN: 1573-8469
    Keywords: antibiotic screening ; colony differentiation ; cross-reaction ; detection level ; fluorescent background reduction ; micro colony assay ; miniaturized agar plating ; plating efficiency ; potato heel end
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Various compounds and basal media were tested for their suitability to create a semi-selective medium for isolation ofClavibacter michiganensis subsp.sepedonicus (Cms) from cattle manure slurry containing c. 108 colony forming units (cfu) per ml. Plating efficiency of Cms in yeast glucose mineral medium (YGM) was 104% compared with yeast peptone glucose medium. Nalidixic acid, polymyxin B sulphate and the experimental disinfectant S-0208 inhibited colony growth of cattle slurry bacteria as compared with Cms in YGM. The optimal concentration of these inhibitors in combination was determined by modified agar diffusion tests and by pour plating in 24-well tissue culture plates. The semi-selective medium YGMI consisted of YGM supplemented with nalidixic acid (2 mg/l), polymyxin B sulphate (30 mg/l) and S-0208 (125 mg/l). Plating efficiency varied for Cms between 50.9 and 69.6%, for cattle slurry bacteria between 1.8 and 2.5% and for saprophytes from potato heel end extracts between 11.5 and 27.4%. Differentiation of Cms colonies from other colonies was based on their small and bluish colony morphology in pour plates and on immunofluorescence colony-staining (IFC). IFC of a pure culture of micro colonies of Cms in YGM was possible after one day incubation (colonies c. 5 cells). Green background fluorescence in the agar gels was prevented by addition of Tween 20 (0.1%) to the washing buffer and the use of 1% agar gels. IFC of macro colonies of Cms in YGMI, visible with 4x objective magnification, was possible after 4 days. The detection level of the target organism in artificially inoculated cattle slurry in YGMI based on colony morphology varied between 1.4×103 and 2.3×104 cfu per ml of cattle slurry. Miniaturized plating combined with IFC, using wells in tissue culture plates (Φ=16 mm), proved suitable for detection, but was c. 30 times les sensitive. The recovery of Cms was negatively correlated with the number of saprophytic colonies in the agar plates (R 2=0.74).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01974227
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